RelA-associated inhibitor, process for producing the same and utilization thereof

ABSTRACT

Human RelA-associated inhibitor (RAI) comprises 351 amino acids and is capable of binding to p 65 , a subunit of the transcription factor NFκB. A process for producing RAI, a cDNA encoding it, a fragment capable of hybridizing selectively to the cDNA sequence, a plasmid for expression and duplication comprising the cDNA, a host cell transformed with the plasmid, an antibody against the polypeptide, a pharmaceutical composition comprising the polypeptide or the antibody against it and a method for diagnosis of diseases using the antibody against the RAI are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.09/857,447, filed Jun. 4, 2001 (now U.S. Pat. No. 6,582.933); which is a371 of PCT/JP99/06753, filed Dec. 2, 1999, the disclosure of each ofwhich is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a protein that inhibits activation oftranscription factor NFκB.

More detailed, the present invention relates to p65-binding protein(called RelA-associated inhibitor or abbreviated as RAI hereinafter)that binds to p65, which is a submit of transcription factor NFκB, andthat inhibits transcriptional activity of NFκB, a process for producingit, a cDNA encoding it, a vector comprising the cDNA, a host celltransformed with the vector, an antibody against the inhibitor, and apharmaceutical composition comprising the inhibitor or the antibody.

Further, the present invention relates to recombinant production ofthese proteins (especially, in vivo production), a nucleotide encodingthem, a vector for expression and duplication and treatment and/orprevention of adult respiratory distress syndrome (ARDS), asthma,Allograft rejection, inflammatory diseases (inflammatory arthritis,angitis etc.), ischemic diseases, autoimmune diseases including chronicrheumatism, metastasis and invasion of cancer, vasoreconstriction anddiseases induced by NFκB besides above.

TECHNICAL BACKGROUND

An acceleration of expressing many and various genes is observed ininflammation. Such genes include ones encoding interleukin,transcription factors, cohesive molecules, and factors in coagulationsystem etc. NFκB which is a transcription factor has been said to relateto the transcription of such genes mostly.

It has been known that transcription factor NFκB is expressed incytoplasm. The transcription and induction of gene due to NFκB-likeproteins may be caused by activation of the protein. By such anactivation, it becomes possible to translocate the transcription factorprepared in advance from cytoplasm to nucleus.

It is known that this translocation is controlled by phosphorylation anddegradation of suppressor protein which is called for IκB.

Transcription factor NFκB was isolated from matured B cells in the formof binding to 10 nucleotide sequence mochief in κ light chain enhancerfor the first time. Therefore, NFκB was thought to be specific for thegenerating stage of matured B cells. However, NFκB-like proteins havebeen identified in a lot of cells, so it is shown that such a factorrelates to induction of gene transcription generally. Such fact has beenconfirmed by functional identification of an active type NFκB-bindingposition in some inducing genes.

NFκB is a heterodimer consisting of a submit of 50 kDa (p50) and asubmit of 65 kDa (p65).

NF kappa B (NFκB), which is a nuclear factor, is a sequence-specificDNA-binding protein complex which regulates the expression of viralgenomes, including the human immunodeficiency virus (HIV), and a varietyof cellular genes, particularly those involved in immune andinflammatory responses. The members of the NFκB family in mammaliancells include the proto-oncogene c-Rel, p50/p105 (NFκB1), p65 (RelA),p52/p100 (NFκB2), and RelB etc.

All of these proteins share a conserved 300 amino acid region ofhomology known as the Rel homology domain (RHD), which is responsiblefor DNA binding, dimerization, and nuclear translocation of NFκB.

In most cells, Rel family members can form hetero- and homodimers withdistinct specificities in various combinations. A common feature of theregulation of transcription factors belonging to Rel family is theirsequestration in the cytoplasm as inactive complexes with a class ofinhibitory molecules known as IKBs. Treatment of cells with a variety ofinducers such as phorbol esters, intedeukin 1, tumor necrosis factor-α(TNF-α), viral infection and many mitogens and cytokines etc. results inthe dissociation of the cytoplasmic complexes and translocation of freeNFκB into the nucleus. The dissociation of the cytoplasmic complexes isknown to be triggered by the phosphorylation and subsequent degradationof the IKB proteins. Such a degradation exposes the nuclear localizationsequence in the remaining NFκB heterodimer, leading to nucleartranslocation and subsequent binding of NFκB to DNA regulatory elementswithin NFκB target genes. The p65 submit is frequently detected in NFκBcomplexes and has a strong transcription activation potential. p65dimerizes with other NFκB family members and activates gene expressionvia its potent transactivation domain.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a novel protein thatinhibits transcriptional activity of NFκB (NFκB inhibitor), a processfor producing it, a cDNA encoding it, a vector comprising the cDNA, ahost cell transformed with the vector, an antibody against theinhibitor, and a pharmaceutical composition comprising the inhibitor orthe antibody.

The present inventor et al. have thought that novel inhibitors may existbesides IκB which was known as an NFκB inhibitor. From the result offocusing their attention on and an extensive studies of NFκB, especiallyp65 submit, the present inventor et al. have achieved to find out anovel NFκB inhibitor, confirmed amino acid sequence, a cDNA sequnceencoding it, function of the inhibitor and tissue-distribution etc. andthen completed the present invention.

RelA-associated inhibitor of the present invention is regarded asnucleus factor and inhibits NFκB-dependent transcription activation byinhibiting DNA-binding activity of NFκB. The gene encodesRelA-associated inhibitor is a gene having a high homology to theC-terminal region of 53BP2 containing four consecutive ankyrin repeatsand an SH3 domain.

RAI appeared to locate in nucleus with the same location pattern ofTNF-α-induced p65. In addition, RAI inhibits activation of NFκB, butdoes not show any influence on p53-dependent transcription activation inthe cells which is transformed temporarily. Therefore, RAI is a novelp65-binding protein which relates to another mechanism of NFκB-dependenttranscription regulation. The present inventor has confirmed theseinteraction in vitro with bacterially expressed fusion proteins and invivo using immunoprecipitation/Western blot assay.

In spite of its similarity to 53BP2, RAI did not shown any interactionwith p53 in a yeast two-hybrid assay. The cDNA encodes this protein hasa high structual homology to 200 amino acid at the C-terminal region of53BP2 containing four ankyrin repeats and an SH3 domain that areimportant for protein-protein interactions.

In human, mRNA of RAI was specifically expressed in heart, placenta andprostate while it was markedly reduced in liver, skeletal muscle andperipheral blood leukocyte.

Recent rapid developments in techniques for constructing cDNAs andsequencing techniques have made it possible to quickly sequence a largeamount of cDNAs. By utilizing these techniques, a process, whichcomprises constructing cDNAs library from various cells or tussles,cloning cDNAs at random, identifying the nucleotide sequences thereof,expressing novel polypeptides encoded by them to analyze itsphysiological function, is now in progress. Method of yeasttwo-hybridization has been known as one of such techniques.

By this technique, it becomes possible to identify a large amount ofhomologous proteins at the same time and to obtain the aimed proteinonly easily by inserting reporter genes such as fluorescent marker etc.

The present inventor has carried out experiments of such a yeasttwo-hybrid screen using NFκB protein submit p65 as a probe to find anovel factor that binds to a novel p65 and that inhibits an activity ofp65 and then found out the factor of the present invention.

The sequence of RelA-associated inhibitor of the present invention hasbeen confirmed to be novel according to a homology search. Only asequence of RelA-associated inhibitor of the present invention is anovel one among some clones obtained by the process for the sequence ofthe present invention described hereinafter.

The amino acid sequence of RelA-associated inhibitor of the presentinvention is as shown in SEQ ID NO. 1. This amino acid sequnce containsfour ankyrin repeats and an SH3 domain, so it is regarded as a member ofRel family.

The present invention relates to

-   (1) a RelA-associated inhibitor comprising amino acid sequence shown    in SEQ ID NO. 1,-   (2) a cDNA encoding the RelA-associated inhibitor described above    (1),-   (3) a cDNA comprising a nucleotide sequence shown in SEQ ID NO. 2,    and-   (4) a cDNA comprising a nucleotide sequence shown in SEQ ID NO. 3.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the results of analyses of GST and GST-RAI fusion proteins,which were expressed in E. coli, on SDS-PAGE with CBB staining (lanes 1and 2) and analyses of the said proteins by autoradiography afterprotting labeled p65 (lanes 3–5). One tenth of amount of labeled p65 atlane 5 was used at lane 3.

FIG. 2 shows the results of Northern blot analyses of RAI mRNAexpression in human multiple tissues. The lower shows an amount ofβ-actin expression.

FIG. 3 shows a dose-dependent inhibition manner of RAI on NFκB-dependenttranscription activation. The activity of activating p65-inducedtranscription in case of non-expression of RAI is represented byluciferase activity at lane 9. Such an action is inhibited by RAIexpression in a dose-dependent manner (lane 11, lanes 13 and 15).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a RelA-associated inhibitor comprisingan amino acid sequence shown in SEQ ID NO. 1 in substantially purifiedform, a homologue thereof, a fragment of the sequence and a homologue ofthe fragment.

Further, the present invention relates to cDNAs encoding the aboveRelA-associated inhibitor. More particularly, the present inventionrelates to cDNAs comprising nucleotide sequences shown in SEQ ID NOS. 2or 3 and cDNA containing a fragment capable of hybridizing selectivelyto nucleotide sequences shown in SEQ ID NOS. 2 or 3.

A RelA-associated inhibitor of the present invention comprising an aminoacid sequence shown in SEQ ID NO. 1 in substantially purified form willgenerally comprise the polypeptide in a preparation in which more than90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is thatof the SEQ ID NO. 1.

A homologue of RelA-associated inhibitor of the present inventioncomprising an amino acid sequence shown in SEQ ID NO. 1 will begenerally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% homologous to the polypeptide comprising thesaid amino acid sequence over a region of at least 20, preferably atleast 30, for instance 40, 60 or 100 more contiguous amino acids. Such apolypeptide homologue will be referred to a RelA-associated inhibitor ofthe present invention.

In addition, a fragment of RelA-associated inhibitor of the presentinvention comprising an amino acid sequence shown in SEQ ID NO. 1 or itshomologues will be at least 10, preferably at least 15, for example, 20,25, 30, 40, 50 or 60 amino acids in length.

A cDNA capable of selectively hybridizing to the cDNA comprisingnucleotide sequences shown in SEQ ID NOS. 2 or 3 will be generally atleast 70%, preferably at least 80 or 90% and more preferably at least95% homologous to the cDNA comprising the said nucleotide sequence overa region of at least 20, preferably at least 30, for instance 40, 60 or100 or more contiguous nucleotides. Such a cDNA will be referred to “acDNA of the present invention”. A cDNA capable of selectivelyhybridizing to the cDNA also includes cohesive sequence. The conditionfor hybridizing is preferably stringent one.

Fragments of the cDNA comprising nucleotide sequences shown in SEQ IDNOS. 2 or 3 will be at least 10, preferably at least 15, for example,20, 25, 30 or 40 nucleotides in length, and will be also referred to “acDNA of the present invention” as used herein.

A further embodiment of the present invention provides replication andexpression vectors carrying cDNA of the present invention. The vectorsmay be, for example, plasmid, virus or phage vectors provided with anorigin of replication, optionally a promoter for the expression of thesaid cDNA and optionally a regulator of the promoter. The vector maycontain one or more selectable marker genes, for example, a luciferaseexpression gene or an ampicillin resistance gene. The vector may be usedin vitro, for example, of the production of RNA corresponding to thecDNA, or used to transfect a host cell.

A further embodiment of the present invention provides host cellstransformed with the vectors for the replication and expression of thecDNA of the present invention, including the cDNA comprising nucleotidesequences shown in SEQ ID NOS. 2 or 3 or the open reading frame thereof.The cells will be chosen to be compatible with the vector and may forexample, be bacterial, yeast, insect cells or mammalian cells.

A further embodiment of the present invention provides a process forproducing a RelA-associated inhibitor which comprises culturing hostcells of the present invention under conditions effective to express aRelA-associated inhibitor of the present invention. Preferably, inaddition, such a process is carried out under conditions in which thepolypeptide of the present invention is expressed and then produced fromthe host cells.

cDNA of the present invention may also be inserted into the vectorsdescribed above in an antisense orientation in order to prove for theproduction of antisense. Such antisense may be used in a method ofcontrolling the levels of a polypeptide of the present invention in acell.

The invention also provides monoclonal or polyclonal antibodies againsta RelA-associated inhibitor of the present invention. The inventionfurther provides a process for the production of monoclonal orpolyclonal antibodies to the RelA-associated inhibitor of the presentinvention. Monoclonal antibodies may be prepared by common hybridomatechnology using RelA-associated inhibitor of the present invention orfragments thereof, as an immunogen. Polyclonal antibodies may also beprepared by common means which comprise inoculating host animals, (forexample, a rat or a rabbit etc.), with RelA-associated inhibitor of thepresent invention and recovering immune serum.

The present invention also provides pharmaceutical compositionscomprising a RelA-associated inhibitor of the present invention, or anantibody thereof, in association with a pharmaceutically acceptablediluent and/or carrier.

The present invention also provides a method for diagnosis of diseasesetc. induced by NFκB using an antibody against a RelA-associatedinhibitor of the present invention. The various methods for diagnosisusing such an antibody have been known. For example, it may be carriedout by determination of amount of enzyme according to EIA method etc.using an antigen-antibody reaction.

The RelA-associated inhibitor of the present invention specified in (1)includes that which a part of their amino acid sequence is lacking(e.g., a polypeptide comprised of the only essential sequence forrevealing a biological activity in amino acid sequence shown in SEQ IDNO. 1), that which a part of their amino acid sequence is replaced byother amino acids (e.g., those replaced by amino acid having a similarproperty) and that which other amino acids are added or inserted into apart of their amino acid sequence, as well as those comprising the aminoacid sequence shown in SEQ ID NO. 1.

As known well, there are one to six kinds of codon as that encoding oneamino acid (for example, one kind of codon for Methionine (Met), and sixkinds of codon for Leucine (Leu) are known). Accordingly, the nucleotidesequence of cDNA can be changed in order to encode the polypeptidehaving the same amino acid sequence.

The cDNA of the present invention, specified in (2) includes a group ofevery nucleotide sequence encoding RelA-associated inhibitor in (1)shown in SEQ ID NO. 1. There is a probability that yield of aRelA-associated inhibitor is improved by changing a nucleotide sequence.

The cDNA specified in (3) is the embodiment of the cDNA shown in (2),and indicate the sequence of natural form.

The cDNA shown in (4) indicates the sequence of the cDNA specified in(3) with natural non-translational region.

(5) Sequnces shown in SEQ ID NO. 4 reveal the correspondence betweencDNA sequence in (4) shown in SEQ ID NO. 3 and amino acid sequence shownin (1).

cDNA carrying nucleotide sequence shown in SEQ ID NO. 3 andRelA-associated inhibitor comprising amino acid sequnce shown in SEQ IDNO. 1 of the present invention may be prepared by the following method:

-   1) Identification of the protein capable for interacting on p65 may    be carried out by yeast two-hybridization method.

The central part of p65 (possessing properties comprising a dimerdomain, nucleus translocatin signal and multiple proline) is cloninginto plasmid (pAS2-1 etc.) to contain yeast GAL4-binding domain in thesame frame.

-   2) Human (placenta, brain etc.) cDNA library constructed in GAL4    transcription activating domain vector pACT2 is cloned using the    obtained plasmid as a probe.

The yeast tranformats are obtained by screening using yeast (Y190 strainetc.). From them, clones which are incubated on the culture forselection are screened according to blue-colouring byfilter-lift-glactosidase assay.

-   3) Rescreening is carried out using alternative yeast (Y187 strain    etc.) to confirm plasmid clone capable for interacting p65 probe    specifically. It is possible to identify these clones according to    both strands DNA sequencing.-   4) The clones encoding RelA-associated inhibitor of the present    invention may be obtained from the obtained clones by removing known    ones.-   5) Aimed full-length cDNA of about 6 kbp may be isolated by    plaque-hybridization using the obtained DNA fragment from human    library (placenta, brain etc.).-   6) RelA-associated inhibitor of the present invention may be    prepared from the isolated cDNA using expression cells (CHO cell,    COS cell etc.) according to known methods.

Apart from the ankyrin repeats and SH3 domain, the RAI sequence isunrelated to that of any protein in current databases using BLASTresearch.

It is necessary to confirm whether the full-length or almost full-lengthcDNA has been obtained. It has been confirmed that the obtained cDNA isalmost full-length by Northern blot analysis according to comparisonwith size of the obtained mRNA.

Once the nucleotide sequences shown in SEQ ID NOS. 2 and 3 aredetermined, cDNAs of the present invention are obtained by chemicalsynthesis, or by hybridization making use of nucleotide fragments whichare chemically synthesized as a probe. Furthermore, cDNAs of the presentinvention may be obtained in desired amount by transforming a vectorthat contains the cDNA into a proper host, and culturing thetransformant.

The polypeptides of the present invention may be prepared by:

-   (1) isolating and purifying from an organism or a cultured cell,-   (2) chemically synthesizing, or-   (3) using recombinant DNA technology etc., preferably, by the method    described in (3) in an industrial production.

Examples of expression system (host-vector system) for producing apolypeptide by using recombinant DNA technology are the expressionsystems of bacteria, yeast, insect cells and mammalian cells.

In the expression of the polypeptide, for example, in E. Coli, theexpression vector may be prepared by adding the initiation codon (ATG)to 5′ end of a cDNA encoding mature peptide, connecting the cDNA thusobtained to the downstream of a proper promoter (e.g., trp promoter, lacpromoter, λ PL promoter, T7 promoter etc.), and then inserting it into avector (e.g., pBR322, pUC18, pUC19 etc.) which functions in an E. Colistrain.

Then, an E. Coli strain (e.g., E. Coli DH5α strain, E. Coli JM109strain, E. Coli HB101 strain, etc.) which is transformed with theexpression vector described above may be cultured in an appropriatemedium to obtain the desired polypeptide. When a signal peptide ofbacteria (e.g., signal peptide of pel B) is utilized, the desiredpolypeptide may be also released in periplasm. Furthermore, a fusionprotein with other polypeptide may be also produced readily.

In the expression of the polypeptide, for example, in a mammalian cells,for example, the expression vector may be prepared by inserting the cDNAencoding nucleotide sequence shown in SEQ ID NO. 3 into the downstreamof a proper promoter (e.g., SV40 promoter, SRα promoter, LTR promoter,metallothionein promoter etc.) in a proper vector (e.g., retrovirusvector, papilloma virus vector, vaccinia virus vector, SV40 vector,etc.). A proper mammalian cell (e.g., human undifferenciatedeosinophilic leukemia EOL cell, COS-7 cell, CHO cell, mouse L cell etc.)is transformed with the expression vector thus obtained, and then thetransformant is cultured in a proper medium to express the aimedpolypeptides. Thus obtained polypeptides may be isolated and purifiedwith the known biochemical methods.

INDUSTRIAL UTILITY

RelA-associated inhibitor (polypeptides) of the present invention iscapable of binding to p65 in nucleus, which is a submit of transcriptionfactor NFκB, and inhibiting activity of NFκB and transcription of genein nucleus. Therefore, it is useful for treatment and/or prevention ofadult respiratory distress syndrome (ARDS), asthma, Allograft rejection,inflammatory diseases (inflammatory arthritis, angitis etc.), ischemicdiseases, autoimmune diseases (especially, AIDS), metastasis andinvasion of cancer, vasoreconstriction and diseases induced by NFκBbesides the above. Further, it also relates to cardiogenesis andsuppression of cardiac inflammation, so it may be used for regeneratingcardiomuscle cells.

In addition, the antibody of the present invention may be used fordiagnosis of the above diseases.

A pharmaceutical composition comprising RelA-associated inhibitor,antibody against it or antisence thereof of t he present invention andpharmaceutically acceptable diluent and/or a carrier is also included inthe present invention.

RelA-associated inhibitor, cDNA encoding it, antibody against it andantisence of the present invention may be administered as solidcompositions, liquid compositions or other compositions for oraladministration, as injections, liniments or suppositories etc. forparental administration.

Solid compositions for oral administration include compressed tablets,pills, capsules, dispersible powders, and granules. Capsules includesoft or hard capsules.

The doses to be administered depend upon age, body weight, symptom,desired therapeutic effect, route of administration, and duration of thetreatment etc. In human adults, one dose per person is generally between100 μg and 100 mg, by oral administration, up to several times per day,and between 10 μg and 100 mg, by parental administration up to severaltimes per day.

As mentioned above, the doses to be used depend upon various conditions.Therefore, there are cases in which doses lower than or greater than theranges specified above may be used.

The antibody against RelA-associated inhibitor of the present inventionmay be used for diagnosis of the above diseases.

An quantitative assay of RelA-associated inhibitor of the presentinvention in the body may be carried out using polychlonal or monoclonalantibody against it. Based on the assay, it is possible to study therelationship between RelA-associated inhibitor of the present inventionand diseases or diagnose such diseases.

Polyclonal and monoclonal antibodies can be prepared using thepolypeptide of the present invention or its fragment as an antigen byconventional methods. Further, purification or gene cloning of proteins(NFκB) capable of binding to the RelA-associated inhibitor (polypeptide)can be performed using the present polypeptide. In addition, the presentpolypeptide may be used for screening RelA-associated inhibitor agonistor antagonist.

cDNAs of the invention are useful as not only the important andessential template for the production of the RelA-associated inhibitorof the invention which is expected to be largely useful, but also beuseful for diagnosis or therapy (for example, treatment of gene lacking,treatment to stop the expression of the RelA-associated inhibitor byantisense DNA (RNA)). Genomic DNA may be isolated using the cDNA of theinvention, as a probe. As the same manner, a human gene encoding whichcan be highly homologous to the cDNA of the invention, that is, whichencodes a polypeptide highly homologous to the RelA-associated inhibitorof the invention and a gene of animals excluding human which can behighly homologous to the cDNA of the invention, also may be isolated.

BEST MODE CARRYING OUT THE INVENTION

The invention is illustrated by the following Examples and ReferenceExamples, but not limit the invention.

EXAMPLE 1 Construction of Plasmids-Plasmids

Construction of plasmids-plasmids were constructed by standard methods.

The human p65 (amino acids 176–405) fragment was cloned into pAS2-1(Clontech Co., Palo Alto) by the yeast two-hybrid system.

PCR amplification using 5′ and 3′ oligonucleotides containing BamHIrestriction sites, i.e., Forward: 5′-GGCGGATCCCTCCGCCTGCCGCCTGTC-3′ (SEQID NO. 5), and Reverse: 5′-GCTGGATCCGGGGCAGGGGCTGGAGCC-3′ (SEQ ID NO. 6)was carried out. The resulting plasmid was named pAS2-1-p65 (176–405).pACT2-p35, encoding the largest RelA Associated Inhibitor (hereinafterabbreviated as RAI) insert obtained from library screening, was cut withEcoRI and Xhol. This EcoRI-Xhol fragment was purified and cloned intoEcoRI-Xhol-digested vector pGEX-5X-2 (Pharmacia Co.).

RAI cDNA was cloned as described below.

The full length RAI was amplified using the oligonucleotides containingSalI and BamHI restriction sites, i.e., Forward:5′-CAATGGTCGACATGGATCTGACTCTTGCTG-3′ (SEQ ID NO. 7) and Reverse:5′-GATCAGGATCCTCAGGCCAAGCTCCTTTGT-3′ (SEQ ID NO. 8) respectively, andcloned as a SalI-BamHI fragment into plasmid pEGFP-C1 to obtain plasmidpEGFP-RAI which may produce fusion protein with RAI in frame with GFP.

pEGFP-RAI (1–146) was prepared from pEGFP-RAI by cutting with BssHII andBamHI, blunt ended with Klenow enzyme and cloning into pEGFP-C1.

The pEGFP-RAI (132–351) was prepared by amplifying the corresponding RAIfragment by PCR using the oligonucleotides F132 containing EcoRI andBamHI sites, ie., Forward: 5′-GCTTCGAAT TCTGTGCTGCGGAAGGCG-3′ (SEQ IDNO. 9) and Reverse: 5′-GATCAGGATCCTCAGGCCAAGCTCCTTTGT-3′ (SEQ ID NO. 10)respectively, and by cloning into pEGFP-C1. pFLAG-CMV-RAI was preparedby inserting the full-length RAI fragment from pEGFP-RAI into Sall andBamHI restriction sites of pFLAG-CMV-2 vector.

All the PCR were carried out using Expand high fidelity system(Boehringer Mannheim Co.).

All the constructs were confirmed by dideoxynucleotide sequencing usingABI PRISM dye terminator cycle sequencing ready reaction kit (PerkinElmer Co.).

Plasmids PG13-Luc containing a generic p53 response element, andpCMV-p53wt expressing wild type human p53, were generous gifts from Dr.B. Vogelstein. The constructions of 4kBw-Luc and 4kBm-Luc have beencarried out by known methods described in articles.

Yeast two-hybrid screen—pAS2-1-p65 (176–405) was used as probe to screenhuman placenta and brain cDNA libraries according to the MatchmakerTwo-Hybrid System protocol (Clontech Co.). Positive yeast clones wereselected by prototrophy for histidine and by expression ofβ-galactosidase. Library derived from plasmids were rescued frompositive clones and transformed into E. coli HB101.

Subsequent two-hybrid assays were carried out by introducing theplasmids into yeast strain Y187 carrying a Gal1-regulated reporter geneand detected the expression of β-galactosidase to confirm the specificbinding.

Plasmids containing cDNA clones that specifically interact with p65(176–405) were identified by restriction mapping, PCR using genespecific primers, and DNA sequencing of both strands.

Sequence Network BLAST searches were conducted by using the NationalCenter for Biotechnology Information (NCBI) on-line service.

EXAMPLE 2 Culturing Cell and Transfection

HeLa, 293T and Saos-2 and COS-1 cells were incubated at 37° C. inDulbecco's modified Eagle's medium with 10% heat-inactivated fetalbovine serum (FBS), 1 mM glutamate, 100 U/ml penicillin, and 100 mg/mlstreptomycin. Jurkat human T cells were maintained in RPMI 1640 with 10%FBS plus antibiotics.

Cells were transfected using SuperFect transfection reagent (Qiagen Co.)according to the manufacturer's recommendations.

EXAMPLE 3 Purification and Expression of Recombinant Proteins

pGEX-5X-2 plasmid encoding glutathione S-transferase (GST)-fusionproteins were transformed in E. coli strain BL21 (DE3) pLysS followinginduction with isopropyl-1-thio-β-D-galactopyranoside (IPTG, 0.1 mM) at28° C. overnight. Recombinant GST-fusion proteins were purified byincubating the bacterial extracts in buffer A (Tris-HCl (pH 7.4; 50 mM),DTT (1 mM), EDTA (1 mM), EGTA (0.1 mM), NaCl (100 mM), benzamidine (1mM), phenylmethylsulphonyl fluoride (PMSF) (0.1 mM),tosylphenylalanylchloromethylketone (0.1 mM), 0.25% (v/v) Nonidet P-40(NP-40)) with glutathione-sepharose beads (Pharmacia Biotech Co.).

The beads were washed five times with ice-cold buffer A and suspended inbuffer A (1 ml). The purification and examination of quantity of boundGST fusion proteins were carried out by SDS-PAGE using 5–20% sodiumdodecyl sulfate-polyacrylamide and by stain using Coomassie brilliantblue.

EXAMPLE 4 In Vitro Assays

³⁵S-methionine-labeled p65 was synthesized by using the TNT T7/SP6 wheatgerm extract coupling system (Promega Co.) according to themanufacturer's protocols.

For in vitro protein-protein interaction studies, an equal amount of thein vitro-translated ³⁵S-methionine-labeled p65 was incubated with 5 mgof purified GST-RAI fusion proteins or GST alone (as a negative control)that were bound to glutathione-sepharose beads at 25° C. for 1 h inbuffer A (250 ml).

The beads were washed by resuspension and centrifugation five times with1 ml of ice-cold binding buffer A containing 0.1% NP-40. Bound proteinswere eluted with an equal volume of SDS loading buffer, boiled for 3min, dissolved into 5–20% SDS-PAGE, and visualized by autoradiography.

EXAMPLE 5 Isolation of Full-Length RAI

A fragment encoding the longest RAI obtained from yeast two-hybridscreen was used as a hybridization probe to screen a human placenta5′-STRETCH PLUS cDNA library (Clontech Co.). The probe was labeled by³²P using Prime-It Random Primer Labeling Kit (Stratagene).Hybridization was performed according to the manufacturer'srecommendations. The filters were washed to a final stringency of0.1×SSC-0.5% SDS at 65° C. and exposed to X-ray film (X-Omat AR; EastmanKodak Co.) overnight with intensifying screens. The films were developedand plaques hybridizing on filters were identified and isolated. Phageswere eluted from agarose plugs in SM buffer and stored at 4° C. (primaryplaque pools). Secondary and tertiary plaque purifications wereperformed in similar fashion to that for the primary pools and a seriesof such procedures was repeated until single plaques could be isolated.

EXAMPLE 6 Western Blotting

Full-length RAI was cloned into a CMV promoter expression vectordesigned to place a FLAG epitope tag at the 5′ end of the open readingframe. 293T cells were transfected using SuperFect transfection reagentas described above. Proteins were isolated from extraction of wholecells in a SDS-containing buffer. Western blotting was performed by astandard technique, using anti-FLAG antibody (Santa Cruz Co.) at adilution of 1/1,000. Secondary antibody, horseradishperoxidase-conjugated anti-rabbit IgG antibody was used at a dilution of1/2,500, and protein bands were visualized by enhancedchemiluminesacence (Amersham Co.).

EXAMPLE 7 Co-Immunoprecipitation

After transfection, 293T cells were cultured for 24 hour and thenharvested (proliferated). After washing with PBS, cells were lysed in350 ml ice-cold lysis buffer (Tris HCl (pH 7.4; 50 mM), 150 mM NaCl, 2mM EDTA, 1 mM PMSF, 1 mM DTT, 0.2% NP-40, 10 mM NaF, 10μg/ml aprotinin,10 by centrifugation. The supernatants were incubated with anti-FLAG M2murine monoclonal antibody (Kodak Co.) or control mouse monoclonalantibody (Dako Co.) or rabbit polyclonal IκB-α antibody (Santa Cruz Co.)overnight at 4° C. and then with protein A-Sepharose (Pharmacia Co.) for4 h. The beads were washed six times with 1 ml lysis buffer. Boundproteins were eluted with an equal volume of SDS loading buffer andresolved on 10% SDS-PAGE. Western blot was conducted as described aboveusing anti-NF-κB p65 (C-20) antibody (Santa Cruz Co.).

EXAMPLE 8 Northern Blot Hybridization

Northern blot hybridization of the human multiple tissue blots (ClontechCo.) was performed according to the manufacturer's recommendations. Afull-length of RAI cDNA was used as a probe. The blots were also probedwith an β-actin fragment to confirm that equal amount of RNAs wasblotted.

EXAMPLE 9 Microscopic Examination

HeLa cells were cultured in 4-well chamber slides and transfected withplasmids expressing various GFP fusion proteins using SuperFecttransfection reagent (Qiagen Co.). After 24 h, cells were fixed byreacting in 4% paraformaldehyde for 15 min and stained with theDNA-binding dye Hoechst-33342 at room temperature for 15 min followed bywashing in phosphate-buffered saline (PBS). The intracellular locationswere examined by fluorescence microscopy.

EXAMPLE 10 Immunofluorescence Microscopy

pEGFP-RAI (0.1 mg) was transfected into HeLa cells cultured in 4-wellchamber slides. Twenty-four hour after transfection, cells wereuntreated or treated with 10 ng/ml TNF-α, then were immuno-stainedaccording to the methods described as hereinbefore. Briefly, cells werefixed with 4% (w/v) paraformaldehyde/PBS for 10 min at room temperatureand then were permeabilized by 0.5% Triton X-100/PBS for 20 min at roomtemperature. They were then incubated with rabbit polyclonal antibodyagainst p65 (Santa Cruz Co.) for 1 h at 37° C. After washing with PBS,the cells were incubated with TRITC-conjugated goat anti-rabbit IgGantibody (Cappel Organon Teknika Co.) for 30 min at 37° C. Then thecells were stained with Hoechst-33342 at room temperature for 15 min toview the nuclear morphology. The slides were examined by fluorescencemicroscopy.

EXAMPLE 11 Luciferase Assay

HeLa and Saos-2 cells were cultured in 12-well plates and transfectionswere performed with SuperFect transfection reagent (Qiagen Co.). Foreach transfection, 50 ng of KB-dependent or mutant reporter plasmid(4kBw-Luc or 5kBm-Luc) or 250 ng of p53-dependent luciferase reporterplasmid (PG13-Luc) and 30 ng of internal control plasmid pRL-TK wereused. The empty vector pEBVHisB or pFLAG-CMV-2 was used to adjust thetotal amount of DNA transfected to 1.5 μg. Twenty-four hourspost-transfection, cells were harvested to measure the luciferaseactivity (Sato, T et al, AIDS Res. Hum. retrovir. 14, 293–298 (1998)).Jurkat cells were transfected with indicated plasmids. Twenty-four hourpost-transfection, the cells were induced by 5 ng/ml TNF-α for 24 h andharvested for luciferase assay.

EXAMPLE 12 Gel Electrophoretic Mobility Shift Assay

293T cells were transfected for 24 h and nuclear extracts were preparedaccording to the known methods described previously (Okamoto, T, et al,Virology 177, 606–614 (1990)). The nuclear pellet was lysed in 35 ml ofextraction buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 350 mM NaCl, 0.1 mMEDTA, 1 mM DTT, 0.1 mM PMSF, 10% glycerol).

The protein content was measured by the DC protein Assay (Bio-RAD Co.).The gel electrophoretic mobility shift assay (EMSA) was performedaccording to the known methods described previously (Yang, J. -P., etal, FEBS Lett. 361, 89–96 (1995): Liu, Z. -Q., et al, FEBS Lett. 385,109–113 (1996)) using the kB sequence taken from the humanimmunodeficiency virus LTR. As a control, Oct-consensus olignucleotide:5′-TGTCGAATGCAAATCACTAGAA-3′ (SEQ ID NO. 11) was used. Oligonucleotideswere labeled using DNA polymerase Klenow fragment (Takeda BiomedicalsCo.) and [α-³²P]dATP (3000 Ci/mmol, ICN Pharmaceuticals Inc.).

DNA binding reactions were performed at room temperature for 15 min in10-μl reaction voluminous containing 5 μg of nuclear extracts. Analysisof binding complexes was performed by electrophoresis in 5% nativepolyacrylamide gels with 0.5×tris-borate-EDTA buffer, followed byautoradiography. For DNA competition experiments, unlabeled competitoroligonuclotides were added into the reaction mixture at 50-fold molarexcess over the probe.

RESULTS Isolation of RAI cDNA Clones as a p65-Binding Protein

The yeast two-hybrid system has been employed to identify proteins thatinteract with p65. DNA fragment encoding amino acids (176–405) of p65,which contained the dimerization domain, nuclear localization signal andthe proline-rich motif, was cloned into pAS2-1 containing a DNA-bindingdomain of yeast GAL4 in frame. The resulting plasmid, pAS2-1-p65(176–405), was used as a probe to screen human cDNA libraries (placentaand brain) that had been constructed in GAL4 transcriptional activationdomain vector pACT2. The yeast strain Y190 was used for screening. Fromapproximately 1.56×10⁶ Y190 yeast transformants, 78 colonies grew onselective medium and turned blue when tested in a filter liftgalactosidase assay.

Resulting from re-screening using Yeast strain Y187, 63 of plasmidclones encoding the proteins which specifically interacted with the p65bait were found. These clones were then characterized by DNA sequenceanalysis of both strands.

Primary nucleotide sequencing revealed that in addition to those alreadyidentified as p65-binding proteins, such as genes encoding IκB-β/Trip9(4 clones), p50/p105 (3 clones), p65/RelA (2 clones), c-Rel (1 clone)and 53BP2 (2 clones), four clones of an unidentified gene encoding aprotein not previously described in the GenBank database were isolated(all of them were derived from human placenta cDNA library).

The present inventor has called this new protein RelA AssociatedInhibitor (RAI). These four clones identified by the two-hybrid screenshared the same 3′ end, but contained different insert sizes.

To confirm the direct interaction between p65 and RAI, in vitro bindingassay between recombinant RAI and p65 proteins was performed. The cDNAinsert from plasmid pACT2-RAI was cloned in frame into the GSTexpression plasmid for production of the GST-RAI fusion protein.

The GST fusion proteins were expressed in E. coli and purified byglutathione-sepharose beads (FIG. 1, lanes 1 and 2).

[³⁵S]methionine-labeled p65 was synthesized by in vitro transcriptionand translation protocol using wheat germ extract (FIG. 1, lane 3).

The purified GST-fusion proteins bound to glutathione-sepharose beadswere incubated with the in vitro synthesized p65 protein. The beads werewashed and bound material was analyzed by SDS-PAGE followed byautoradiography. As shown in FIG. 1, p65 interacted strongly with RAI(lane 5). Binding between p65 and RAI was considered as specific becauseno interaction of p65 was observed with the control GST protein (lane4).

EXAMPLE 13 Isolation of Full-Length RAI

The four independent clones isolated from the yeast two-hybrid screenwere partial-length cDNA derived from the same gene. Plaquehybridization of a human placenta cDNA library with the partial cDNAfragment of RAI isolated from the two-hybrid screen was performed toobtain a full-length clone. About 10⁶ plaques were screened. Nine cDNAclones were isolated with approximately 1 kb to 6 kb in length. Twolongest cDNAs were sequenced on both strands and were found to containthe complete open reading frame (ORF) for RAI. The clones differed attheir 5′ end, and each contained all of the sequences present inpACT2-RAI. Both contained Alu repeat element in their 5′ untranslatedregions. The likely translation initiation codon of RAI was preceded byan in-frame stop codon located 27 bp upstream. Although the nucleotidesequence 5′ of the first ATG (CTGGCGATG) does not resemble the consensusinitiation sequence, the present inventor designated this ATG as theputative translation start site. DNA sequence analysis revealed an openreading frame predicted to encode a putive protein of 351 amino acidresidues with a predicted molecular mass of 40 kDa. Interestingly, aBLAST search revealed that the C-terminal of this protein has extensivehomology to the C-terminal region of 53BP2, previously reported as p53binding protein in a yeast two hybrid screen (Iwabuchi, K. et al, Proc.Natl. Acad. Sci. USA 91, 6098–6102 (1994)).

RAI protein was predicted to contain four consecutive ankyrin repeats(145 Arg-174 Asn, 176 Pro-207 Ala, 209 Val-241 Ala, 243 Phe-275 Glu) andan SH3 domain (287 Tyr-388 Phe) at its C-terminus as that of 53BP2, with52% amino acid identity with 53BP2 at the C-terminal of 223 amino acidresidues.

Apart from the ankyrin repeats and SH3 domain, the RAI sequence isunrelated to that of any protein in current databases using BLASTresearch.

EXAMPLE 14 Tissue Distribution of RAI in Human Tissues

To determine the expression pattern of RAI, present inventor performed aNorthern blot analysis on a human multi-tissue blot. As shown in FIG. 2,the RAI mRNA was detected as an approximately 3.4-kb and 6.0-kb band inmost cases. From FIG. 2 clearly, the expression of RAI mRNA wassignificantly high in human heart, placenta and prostate while it wasmarkedly reduced in liver, skeletal muscle, testis and peripheral bloodleukocyte.

EXAMPLE 15 Inhibitory Action of RAI on NFκB-Dependent Transactivation

Since RAI was shown to bind p65 through its ankyrin repeats and SH3domain as similarly to IκB family proteins, the present inventorconfirmed whether RAI had any effect on the kB-dependent transcriptionalactivation. The luciferase reporter plasmid containing four tandemcopies of the kB sequence was co-transfected into 293 T cells with anexpression vector for FLAG-RAI and/or p65 expression vector. Twenty-fourhours after transfection, luciferase activities were determined. Asshown in FIG. 3, co-transfected p65-expressing plasmid as an activatorstrongly transactivated the reporter gene (FIG. 3, lane 9).Co-transfection with the RAI expressing plasmid resulted in theinhibition of the p65-induced NF KB activity in a dose-dependent manner.Although to a lesser extent, the inhibitory effect of RAI on the basallevel of kB-dependent luciferase expression was observed (FIG. 3, lanes11, 13 and 15).

1. A substantially pure polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:1.